It’s the time of year when Microbiology Registrars begin to have palpitations and panic attacks about the impending FRCPath Part 2 exams in spring. All Microbiology Consultants in the UK (and also many overseas) have done the exams to attain Fellowship of the Royal College of Pathologists, and it is a gruelling experience. I have had the pleasure of helping a number of Registrars prepare for the exam and this year is no different. For those of you who don’t know, the FRCPath Part 2 exam is four days long: day 1 is six hours of written exams and days 2-4 is a practical laboratory-based diagnostic exam where the results from day 1 informs the actions of days 2 and 3 subsequently. Getting it wrong can cause spiralling errors...

Is it me or are Microbiology Registrars getting younger?

The first aspect of diagnostic microbiology I get the Registrars to start with is Gram staining and this got me thinking. In the age of automation, MaldiTOF and molecular diagnostics, does Gram’s stain still have a role in the microbiology laboratory? Why do I spend so much time teaching Registrars to become proficient at it? How does it help them in the FRCPath exams and is it still relevant in day-to-day clinical microbiology?

What is it and how does it work? In 1884 a Danish scientist working in Berlin developed a method for distinguishing different types of bacteria viewed with a microscope. His name was Hans Christian Gram, and his technique is still in use in Microbiology laboratories today; the Gram’s stain.

In principal, Gram’s method for staining bacteria is pretty simple. Once the bacteria have been fixed to a glass slide by gentle heat (which stops them being washed off the slide when the staining liquids are added) there are only four main steps and in total it takes less than five minutes to perform a Gram’s stain.

The slide is flooded with crystal violet for about 30 seconds which enters in to bacterial cells. The crystal violet also forms positively charged particles inside the cell that bind to negatively charged components of the bacteria staining it purple.

Iodine is added to the slide for a further 30 seconds which binds to the intracellular crystal violet forming large particles; these become trapped inside the bacterial cell.

Ethanol or acetone are used to wash the crystal violet out of the bacterial cell. Gram-positive bacteria have a much thicker peptidoglycan cell wall and retain the purple stain, whereas Gram-negative bacteria lose their outer cell membrane and their thin cell wall is insufficient to retain the stain and they decolourise. It is critical not to leave the ethanol or acetone on the slide for too long as it will eventually decolour all bacteria, even the Gram-positives! Acetone should only be left on for 2-3 seconds whereas ethanol can be left on for a little longer (up to 10 seconds).

Safranin, neutral red or carbol fuchsin are added for 30 seconds which stain the Gram-negative bacteria red, these can now be seen in contrast to the purple Gram-positive bacteria.

Why do we still do Gram’s stains?Being able to make a Gram’s stain of any bacteria is one of the fundamental skills of a Microbiologist. Almost every identification method and antibiotic sensitivity technique in the laboratory is dependent on being able to say whether a bacterium is Gram-positive, Gram-negative or doesn’t stain with Gram’s method. If the Gram’s stain result is wrong then all the following tests we do in the lab tend to go wrong as well!

If your Gram’s stain is wrong on day one of the FRCPath then it is likely that the rest of the exam is going to go very wrong. Not only will the Gram’s stain appearance help identify the bacterium but it also guides the choice of antibiotics in terms of their activity against Gram-positive bacteria, Gram-negative bacteria, anaerobes and non-culturable bacteria, or any combination of these four.For the FRCPath I even go as far as recommending that Registrars make two slides for their first Gram’s stains in case they are unhappy with the quality of their first attempts or they have an accident e.g. when I did the exam I was so nervous at the beginning that I dropped some of my slides before I had read them and they broke, I pulled myself together, picked up my second set of slides and carried on. Now if I see a registrar is practising without a backup...I consider “accidently” knocking their slides off the bench! Mean huh!?Will Gram’s stain stop being useful in the era of modern diagnostics?Okay I may be an old stick-in-the-mud but I think for the time being Gram’s stains are here to stay. Molecular diagnostics are great, and they can significantly improve patient care but in my opinion they are yet to beat the Gram’s stain for ease of use, cost and speed.

Molecular diagnostics, where a piece of DNA or RNA from a sample is multiplied up, and then detected using other bits of “labelled” RNA or DNA or sequenced to identify the genetic structure of a bacterium, are able to give accurate information about microorganisms, but they are not perfect. Some of the major drawbacks with these techniques are:

Cost – they are expensive often costing hundreds of pounds per test. This prevents their routine use or makes laboratories batch the tests together to try and control the cost in which case tests are often only run once or twice a week making the turnaround time slow

Risk of contamination – because they detect very small amounts of DNA or RNA these tests can easily be contaminated with genetic material which was not actually in the original sample and therefore gives a false positive result

They usually only detect specific microorganisms – most molecular diagnostic tests are specifically targeted at a limited number of microorganisms, and if the patients doesn’t have one of those then the test will give a negative result and the clinicians will be none-the-wiser as to what is going on with the patient

The Gram’s stain doesn’t suffer from any of these drawbacks; it is fast, cheap and none-specific however it is by no means perfect. Compared to molecular diagnostics the results are less specific (you cannot distinguish the subspecies of a bacterium based upon a Gram’s stain), they are less sensitive so small numbers of bacteria can be missed, and they only detect bacteria and fungi (not viruses or parasites). In my opinion, these limitations are minor and therefore a Gram’s stain is still a vital part of laboratory method.For the FRCPath, the Registrars won’t have access to molecular diagnostics, or even MaldiTOF. They will have to rely on conventional microbiological methods, and they don’t get more conventional than Gram’s stain.

Can you tell anything more about a bacterium from a Gram’s stain than just Gram-positive or negative?Different bacteria often have subtly different appearances, and with experience you can tell a lot more from a Gram’s stain than just positive or negative. The table below shows an example of Streptococci, and how they can be distinguished using their Gram’s stain appearance.

Click for larger image

Okay, I accept that not all Gram’s stains can be read to this level, but with experience the vast majority can. If you work in a microbiology laboratory, or are preparing for your FRCPath, have a go at trying to decide what an organism might be on the basis of the Gram’s stain appearance. Test yourself. Let me know how you get on. If you are short of time in the FRCPath being able to narrow down the “further tests” you do could salvage your exam! The future is purple (or maybe red)…So 130 years after its discovery the Gram’s stain with its purple or red blobs is still going strong - it is a fundamental part of the FRCPath Part 2 exam as well as the modern diagnostic microbiology laboratory. If done well it can give huge amounts of information about what a bacterium is and what antibiotics it might be sensitive to. However, if done badly, or misinterpreted, it can cause major confusion and problems both in exams as well as in real clinical scenarios.

I doubt there are many diagnostic tools still in use 130 years after they were first discovered...well... maybe X-rays (originally called roentgenograms after the person who discovered them) and the stethoscope (now considerably changed since the first prototype, it inspires trust in the public even though there is no evidence that it makes any difference to diagnostic accuracy). If you know of any others let me know…

Good luck to all of you preparing for the FRCPath: remember to keep calm, keep it simple, and give good clinical advice…